Bleomycin-induced Pulmonary Fibrosis Model Research Articles

Type II transmembrane serine protease Matriptase is a mediator of pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disorder which remains refractory to therapies. Recently, type II transmembrane serine proteases have emerged as key actors in pathophysiology, by proteolysis of cell surface receptors and/or cell environment, thereby orchestrating cell crosstalks. Among them, deregulation of the matriptase contributes to various proliferative disorders. Hence, we sought to investigate the role of matriptase in pulmlonary fibrosis. Matriptase expression and activity were analyzed in human lung material from controls and IPF patients. Human fibroblasts were stimulated with recombinant matriptase and the activation of key signaling pathways involved in IPF was analyzed. Using the murine model of bleomycin-induced pulmonary fibrosis, mice were administrated for 14 days with 0 or 0.5mg of camostat mesylate, a matriptase inhibitor, and the markers of tissue fibrosis in lung homogenate, and inflammatory cell influx in the bronchoalveolar lavage fluid (BALF) of the animals were assessed. We show that matriptase is upregulated in the lung and BALF of IPF patients compared to controls. In fibroblasts, matriptase induced the activation of p42/44, Akt, and Smad2/3 pathways. Finally, camostat administration in bleomycin-treated mice led to a significant decrease in mortality (39.3% versus 60.4% in bleomycin-treated group). Fibronectin and collagen expression, and inflammatory cell influx, were not significantly different from saline-treated animals in the camostat group.Taken together, matriptase seems instrumental in pulmonary fibrosis, and camostat mesylat could be a promising target for thetreatment of IPF.

Anti-asialo GM1 NK cell depleting antibody does not alter the development of bleomycin induced pulmonary fibrosis.

Despite circumstantial evidence postulating a protective role for NK cells in many fibrotic conditions, their contribution to the development of pulmonary fibrosis has yet to be tested. Lung-migrating NK cells are thought to attenuate the development of bleomycin induced pulmonary fibrosis (BIPF) by providing anti-fibrotic mediators and cytokines, such as IFN-γ. If true, we reasoned that depletion of NK cells during experimentally-induced fibrotic disease would lead to exacerbated fibrosis. To test this, we treated mice with NK cell-depleting antisera (anti-asialo GM1) and evaluated lung inflammation and fibrosis in the BIPF model. While NK cell infiltration into the airways was maximal at day 10 after bleomycin injection, NK cells represented a minor portion (1–3%) of the total leukocytes in BAL fluid. Anti-asialo GM1 significantly abrogated NK cell numbers over the course of the disease. Depletion of NK cells with anti-asialo GM1 before and throughout the BIPF model, or during just the fibrotic phase did not alter fibrosis development or affect the levels of any of the pro-inflammatory/pro-fibrotic cytokines measured (IL-1β, IL-17, IFN-γ, TGF-β and TNF-α). In addition, adoptively transferred NK cells, which were detectable systemically and in the airways throughout BIPF, failed to impact lung fibrosis. These findings indicate that NK cells likely do not play an essential protective role in controlling pulmonary fibrosis development.

BTLA expression on CD4+ T cells negatively regulates the development of pulmonary fibrosis (HUM7P.319)

Abstract The role of the adaptive immune response in idiopathic pulmonary fibrosis (IPF) has been an area of debate. Several studies have shown that patients with IPF had increased numbers of T cells in their lungs, suggesting that these cells may play a role in disease pathogenesis. We have found that decreased expression of an important co-inhibitory molecule, B- and T- lymphocyte attenuator (BTLA) on peripheral blood CD4+ T cells is correlated with IPF patients most likely to rapidly progress in their disease course. Based on these findings, the objective of our study was to determine the role of BTLA on CD4+ T cells in the development of pulmonary fibrosis by utilizing a mouse model of bleomycin-induced pulmonary fibrosis. We found that, similar to our patient studies, bleomycin treated WT mice express decreased levels of BTLA on CD4+ T cells compared to untreated WT mice. BTLA-/- mice exhibited significantly increased weight loss and pulmonary fibrosis compared to WT mice. However, CD4+ depletion in the BTLA-/- mice prior to bleomycin challenge protected these mice, as they lost less weight had decreased lung collagen deposition. These findings support a significant role for the loss of BTLA on CD4+ T cells in the pathogenesis of fibrotic disease progression.

Effect of spiperone on mesenchymal multipotent stromal and hemopoietic stem cells under conditions of pulmonary fibrosis.

The antifibrotic properties of spiperone and its effect on stem and progenitor cells were studied on the model of reversible bleomycin-induced pulmonary fibrosis in C57Bl/6 mice. Spiperone reduced infiltration of the alveolar interstitium and alveolar ducts with inflammatory cells and prevented the growth of the connective tissue in the parenchyma of bleomycin lungs. Apart from anti-inflammatory effect, spiperone suppressed bone marrow hemopoietic cells (CD3, CD45R (B220), Ly6C, Ly6G (Gr1), CD11b (Mac1), TER-119)-, Sca-1+, c-Kit+, CD34- and progenitor hemopoietic cells (granulocyte-erythroid-macrophage-megakaryocytic and granulocyte CFU). Spiperone-induced disturbances of fi brogenesis were paralleled by restoration of endothelial cells in the lung parenchyma, reduction of the number of circulating bone marrow cells and lung mesenchymopoietic cells (mesenchymal multipotent stromal cells (CD31-, CD34-, CD45-, CD44+, CD73+, CD90+, CD106+) and progenitor fi broblast cells), and suppression of multilineage differentiation of multipotent mesenchymal stromal cells (including fi broblast-lineage cells).

Passion fruit peel extract attenuates bleomycin-induced pulmonary fibrosis in mice.

Idiopathic pulmonary fibrosis is a progressive fatal lung disease characterized by excessive collagen deposition, with no effective treatments. We investigated the efficacy of natural products with high anti-inflammatory activity, such as passion fruit peel extract (PFPE), in a mouse model of bleomycin-induced pulmonary fibrosis (PF). C57BL/6J mice were subjected to a single intratracheal instillation of bleomycin to induce PF. Daily PFPE treatment significantly reduced loss of body mass and mortality rate in mice compared with those treated with bleomycin. While bleomycin-induced PF resulted in elevated total numbers of inflammatory cells, macrophages, lymphocytes, and neutrophils in bronchoalveolar lavage fluid on both days 7 and 21, PFPE administration significantly attenuated these phenomena compared with bleomycin group. On day 7, the decreased superoxide dismutase and myeloperoxidase activities observed in the bleomycin group were significantly restored with PFPE treatment. On day 21, enhanced hydroxyproline deposition in the bleomycin group was also suppressed by PFPE administration. PFPE treatment significantly attenuated extensive inflammatory cell infiltration and accumulation of collagen in lung tissue sections of bleomycin-induced mice on days 7 and 21, respectively. Our results indicate that administration of PFPE decreased bleomycin-induced PF because of anti-inflammatory and antioxidant activities.

Histone deacetylase inhibition promotes fibroblast apoptosis and ameliorates pulmonary fibrosis in mice

Idiopathic pulmonary fibrosis (IPF) is a fatal disease, and therapeutic agents have shown only modest efficacy. Epigenetic alterations contribute to the pathogenesis of IPF. The histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), has been approved for clinical use in cancer; however, its potential efficacy in modulating fibroblast survival and lung fibrosis has not been extensively investigated. We investigated the effects of SAHA on apoptosis of primary IPF myofibroblasts and on injury-induced lung fibrosis in a murine model. SAHA-induced apoptosis of IPF myofibroblasts, an effect that was mediated, at least in part, by upregulation of the pro-apoptotic gene Bak and downregulation of the anti-apoptotic gene Bcl-xL. Alterations in the expression of these apoptosis-related genes were associated with histone modifications and changes in DNA methylation. In addition to the expected higher levels of histone acetylation in treated cells, we also detected changes in other histone modifications, such as histone methylation. In a murine model of bleomycin-induced pulmonary fibrosis, SAHA-treated mice displayed decreased lung fibrosis and improved lung function compared to the bleomycin only group. These results suggest that histone deacetylase inhibitors may offer a new therapeutic strategy in IPF by modulating myofibroblast susceptibility to apoptosis.

The Toll-like Receptor 3 L412F Polymorphism and Disease Progression in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a fatal progressive interstitial pneumonia. The innate immune system provides a crucial function in the recognition of tissue injury and infection. Toll-like receptor 3 (TLR3) is an innate immune system receptor. We investigated the role of a functional TLR3 single-nucleotide polymorphism in IPF. To characterize the effects of the TLR3 Leu412Phe polymorphism in primary pulmonary fibroblasts from patients with IPF and disease progression in two independent IPF patient cohorts. To investigate the role of TLR3 in a murine model of pulmonary fibrosis. TLR3-mediated cytokine, type 1 IFN, and fibroproliferative responses were examined in TLR3 wild-type (Leu/Leu), heterozygote (Leu/Phe), and homozygote (Phe/Phe) primary IPF pulmonary fibroblasts by ELISA, real-time polymerase chain reaction, and proliferation assays. A murine model of bleomycin-induced pulmonary fibrosis was used in TLR3 wild-type (tlr3(+/+)) and TLR3 knockout mice (tlr3(-/-)). A genotyping approach was used to investigate the role of the TLR3 L412F polymorphism in disease progression in IPF using survival analysis and longitudinal decline in FVC. Activation of TLR3 in primary lung fibroblasts from TLR3 L412F-variant patients with IPF resulted in defective cytokine, type I IFN, and fibroproliferative responses. We demonstrate increased collagen and profibrotic cytokines in TLR3 knockout mice (tlr3(-/-)) compared with wild-type mice (tlr3(+/+)). TLR3 L412F was also associated with a significantly greater risk of mortality and an accelerated decline in FVC in patients with IPF. This study reveals the crucial role of defective TLR3 function in promoting progressive IPF.

Lysophosphatidic Acid Receptor–2 Deficiency Confers Protection against Bleomycin-Induced Lung Injury and Fibrosis in Mice

Idiopathic pulmonary fibrosis is a devastating disease characterized by alveolar epithelial cell injury, the accumulation of fibroblasts/myofibroblasts, and the deposition of extracellular matrix proteins. Lysophosphatidic acid (LPA) signaling through its G protein-coupled receptors is critical for its various biological functions. Recently, LPA and LPA receptor 1 were implicated in lung fibrogenesis. However, the role of other LPA receptors in fibrosis remains unclear. Here, we use a bleomycin-induced pulmonary fibrosis model to investigate the roles of LPA2 in pulmonary fibrogenesis. In the present study, we found that LPA2 knockout (Lpar2(-/-)) mice were protected against bleomycin-induced lung injury, fibrosis, and mortality, compared with wild-type control mice. Furthermore, LPA2 deficiency attenuated the bleomycin-induced expression of fibronectin (FN), α-smooth muscle actin (α-SMA), and collagen in lung tissue, as well as levels of IL-6, transforming growth factor-β (TGF-β), and total protein in bronchoalveolar lavage fluid. In human lung fibroblasts, the knockdown of LPA2 attenuated the LPA-induced expression of TGF-β1 and the differentiation of lung fibroblasts to myofibroblasts, resulting in the decreased expression of FN, α-SMA, and collagen, as well as decreased activation of extracellular regulated kinase 1/2, Akt, Smad3, and p38 mitogen-activated protein kinase. Moreover, the knockdown of LPA2 with small interfering RNA also mitigated the TGF-β1-induced differentiation of lung fibroblasts. In addition, LPA2 deficiency significantly attenuated the bleomycin-induced apoptosis of alveolar and bronchial epithelial cells in the mouse lung. Together, our data indicate that the knockdown of LPA2 attenuated bleomycin-induced lung injury and pulmonary fibrosis, and this may be related to an inhibition of the LPA-induced expression of TGF-β and the activation and differentiation of fibroblasts.

Antiflammin-1 attenuates bleomycin-induced pulmonary fibrosis in mice

BackgroundAntiflammin-1 (AF-1), a derivative of uteroglobin (UG), is a synthetic nonapeptide with diverse biological functions. In the present study, we investigated whether AF-1 has a protective effect against bleomycin-induced pulmonary fibrosis.MethodsC57BL/6 mice were injected with bleomycin intratracheally to create an animal model of bleomycin-induced pulmonary fibrosis. On Day 7 and Day 28, we examined the anti-inflammatory effect and antifibrotic effect, respectively, of AF-1 on the bleomycin-treated mice. The effects of AF-1 on the transforming growth factor-beta 1 (TGF-β1)-induced proliferation of murine lung fibroblasts (NIH3T3) were examined by a bromodeoxycytidine (BrdU) incorporation assay and cell cycle analysis.ResultsSevere lung inflammation and fibrosis were observed in the bleomycin-treated mice on Day 7 and Day 28, respectively. Administration of AF-1 significantly reduced the number of neutrophils in the bronchoalveolar lavage fluid (BALF) and the levels of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) in the lung homogenates on Day 7. Histological examination revealed that AF-1 markedly reduced the number of infiltrating cells on Day 7 and attenuated the collagen deposition and destruction of lung architecture on Day 28. The hydroxyproline (HYP) content was significantly decreased in the AF-1-treated mice. In vitro, AF-1 inhibited the TGF-β1-induced proliferation of NIH3T3 cells, which was mediated by the UG receptor.ConclusionsAF-1 has anti-inflammatory and antifibrotic actions in bleomycin-induced lung injury. We propose that the antifibrotic effect of AF-1 might be related to its suppression of fibroblast growth in bleomycin-treated lungs and that AF-1 has potential as a new therapeutic tool for pulmonary fibrosis.

Histone Deacetylase Inhibition Downregulates Collagen 3A1 in Fibrotic Lung Fibroblasts

Idiopathic pulmonary fibrosis (IPF) is a deadly disease characterized by chronic inflammation and excessive collagen accumulation in the lung. Myofibroblasts are the primary collagen-producing cells in pulmonary fibrosis. Histone deacetylase inhibitor (HDACi) can affect gene expression, and some, such as suberoylanilide hydroxamic acid (SAHA), are US FDA approved for cancer treatment. In this study, we investigated SAHA’s effects on the expression of collagen III alpha 1 (COL3A1) in primary human IPF fibroblasts and in a murine model of pulmonary fibrosis. We observed that increased COL3A1 expression in IPF fibroblasts can be substantially reduced by SAHA treatment at the level of transcription as detected by RT-PCR; collagen III protein level was also reduced, as detected by Western blots and immunofluorescence. The deacetylation inhibitor effect of SAHA was verified by observing higher acetylation levels of both histone H3 and H4 in treated IPF cells. Chromatin immunoprecipitation (ChIP) experiments demonstrated that the reduced expression of COL3A1 by SAHA is with increased association of the repressive chromatin marker, H3K27Me3, and decreased association of the active chromatin marker, H3K9Ac. In our murine model of bleomycin-induced pulmonary fibrosis, the SAHA treated group demonstrated significantly less collagen III, as detected by immunohistochemistry. Our data indicate that the HDACi SAHA alters the chromatin associated with COL3A1, resulting in its decreased expression.

An Inhibitor of NADPH Oxidase-4 Attenuates Established Pulmonary Fibrosis in a Rodent Disease Model

Idiopathic pulmonary fibrosis is a chronic progressive disease of increasing prevalence for which there is no effective therapy. Increased oxidative stress associated with an oxidant-antioxidant imbalance is thought to contribute to disease progression. NADPH oxidases (Nox) are a primary source of reactive oxygen species within the lung and cardiovascular system. We demonstrate that the Nox4 isoform is up-regulated in the lungs of patients with IPF and in a rodent model of bleomycin-induced pulmonary fibrosis and vascular remodeling. Nox4 is constitutively active, and therefore increased expression levels are likely to contribute to disease pathology. Using a small molecule Nox4/Nox1 inhibitor, we demonstrate that targeting Nox4 results in attenuation of an established fibrotic response, with reductions in gene transcripts for the extracellular matrix components collagen 1α1, collagen 3α1, and fibronectin and in principle pathway components associated with pulmonary fibrosis and hypoxia-mediated vascular remodeling: transforming growth factor (TGF)-β1, plasminogen activator inhibitor-1, hypoxia-inducible factor, and Nox4. TGF-β1 is a principle fibrotic mediator responsible for inducing up-regulation of profibrotic pathways associated with disease pathology. Using normal human lung-derived primary fibroblasts, we demonstrate that inhibition of Nox4 activity using a small molecule antagonist attenuates TGF-β1-mediated up-regulation in expression of profibrotic genes and inhibits the differentiation of fibroblast to myofibroblasts, that is associated with up-regulation in smooth muscle actin and acquisition of a contractile phenotype. These studies support the view that targeting Nox4 may provide a therapeutic approach for attenuating pulmonary fibrosis.

Profibrotic TGFβ responses require the cooperative action of PDGF and ErbB receptor tyrosine kinases

Transforming growth factor β (TGFβ) has significant profibrotic activity both in vitro and in vivo. This reflects its capacity to stimulate fibrogenic mediators and induce the expression of other profibrotic cytokines such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF/ErbB) ligands. Here we address both the mechanisms by which TGFβ induced ErbB ligands and the physiological significance of inhibiting multiple TGFβ-regulated processes. The data document that ErbB ligand induction requires PDGF receptor (PDGFR) mediation and engages a positive autocrine/paracrine feedback loop via ErbB receptors. Whereas PDGFRs are essential for TGFβ-stimulated ErbB ligand up-regulation, TGFβ-specific signals are also required for ErbB receptor activation. Subsequent profibrotic responses are shown to involve the cooperative action of PDGF and ErbB signaling. Moreover, using a murine treatment model of bleomycin-induced pulmonary fibrosis we found that inhibition of TGFβ/PDGF and ErbB pathways with imatinib plus lapatinib, respectively, not only prevented myofibroblast gene expression to a greater extent than either drug alone, but also essentially stabilized gas exchange (oxygen saturation) as an overall measure of lung function. These observations provide important mechanistic insights into profibrotic TGFβ signaling and indicate that targeting multiple cytokines represents a possible strategy to ameliorate organ fibrosis dependent on TGFβ.

Qualitative Rather than Quantitative Changes Are Hallmarks of Fibroblasts in Bleomycin-Induced Pulmonary Fibrosis

Pulmonary fibrosis is characterized by accumulation of activated fibroblasts that produce excessive amounts of extracellular matrix components such as collagen type I. However, the dynamics and activation signatures of fibroblasts during fibrogenesis remain poorly understood, especially invivo. We examined changes in lung tissue cell populations and in the phenotype of activated fibroblasts after acute injury in a model of bleomycin-induced pulmonary fibrosis. Despite clustering of collagen type I-producing fibroblasts in fibrotic regions, flow cytometry-based quantitative analysis of whole lungs revealed that the number of fibroblasts in the lungs remained constant. At the peak of inflammation, fibroblast proliferation and apoptosis were both increased, suggesting that the clustering was not merely a result of proliferation, but also of fibroblast migration from nearby alveolar walls. Parabiosis experiments demonstrated that fibroblasts were not supplied from the circulation. Comprehensive gene expression analysis of freshly isolated fibroblasts revealed a detailed activation signature associated with fibrogenesis, including changes in genes responsible for migration and extracellular matrix construction. The Spp1 gene, which encodes osteopontin, was highly up-regulated and was an identifying characteristic of activated fibroblasts present at the sites of remodeling. Osteopontin may serve as a useful marker of profibrotic fibroblasts. These results provide insights into the cellular and molecular mechanisms underlying pulmonary fibrosis and provide a foundation for development of specific antifibrotic therapies.

Clinical Relevance of Plasma Prostaglandin F2α Metabolite Concentrations in Patients with Idiopathic Pulmonary Fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a devastating lung disease of unknown etiology with few current treatment options. Recently, we determined an important role of prostaglandin F2α (PGF2α) in pulmonary fibrosis by using a bleomycin-induced pulmonary fibrosis model and found an abundance of PGF2α in bronchoalveolar lavage fluid of IPF patients. We investigated the role of PGF2α in human IPF by assessing plasma concentrations of 15-keto-dihydro PGF2α, a stable metabolite of PGF2α.MethodsWe measured plasma concentrations of 15-keto-dihydro PGF2α in 91 IPF patients and compared these values with those of controls (n = 25). We further investigated the relationships of plasma 15-keto-dihydro PGF2α concentrations with disease severity and mortality.ResultsPlasma concentrations of 15-keto-dihydro PGF2α were significantly higher in IPF patients than controls (p<0.001). Plasma concentrations of this metabolite were significantly correlated with forced expiratory volume in 1 second (Rs [correlation coefficient] = −0.34, p = 0.004), forced vital capacity (Rs = −0.33, p = 0.005), diffusing capacity for carbon monoxide (Rs = −0.36, p = 0.003), the composite physiologic index (Rs = 0.40, p = 0.001), 6-minute walk distance (Rs = −0.24, p = 0.04) and end-exercise oxygen saturation (Rs = −0.25, p = 0.04) when patients with emphysema were excluded. Multivariate analysis using stepwise Cox proportional hazards model showed that a higher composite physiologic index (relative risk = 1.049, p = 0.002) and plasma 15-keto-dihydro PGF2α concentrations (relative risk = 1.005, p = 0.002) were independently associated with an increased risk of mortality.ConclusionsWe demonstrated significant associations of plasma concentrations of PGF2α metabolites with disease severity and prognosis, which support a potential pathogenic role for PGF2α in human IPF.

THU0064 Small Animal MRI for Non-Invasive Longitudinal Follow Up of Pulmonary Fibrosis in Mice

BackgroundPulmonary fibrosis, either idiopathic or secondary to diseases such as systemic sclerosis, is a devastating and life threatening disorder for which effective treatment is still lacking. The bleomycin-induced pulmonary fibrosis...

Secreted protein acidic and rich in cysteine (SPARC) is upregulated by transforming growth factor (TGF)-β and is required for TGF-β-induced hydrogen peroxide production in fibroblasts

BackgroundIdiopathic pulmonary fibrosis (IPF) is a poorly understood progressive disease characterized by the recurrent damage of alveolar epithelial cells as well as inappropriate expansion and activation of fibroblasts resulting in pronounced extracellular matrix (ECM) deposition. Although recent studies have indicated the involvement of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein regulating ECM deposition, in the pathogenesis of fibrosis, factors regulating SPARC expression or roles of SPARC in fibrosis have not been fully elucidated.ResultsAmong the profibrotic factors examined in cultured fibroblasts, we showed that SPARC expression was upregulated mainly by transforming growth factor (TGF)-β. We also showed that expression of SPARC in the lung was upregulated in the murine bleomycin-induced pulmonary fibrosis model, which was inhibited by TGF-β receptor I inhibitor. Knockdown of SPARC in fibroblasts using siRNA or treatment with the antioxidant N-acetylcysteine attenuated epithelial cell injury induced by TGF-β-activated fibroblasts in a coculture system. We also demonstrated that SPARC was required for hydrogen peroxide (H2O2) production in fibroblasts treated with TGF-β. Furthermore, TGF-β activated integrin-linked kinase (ILK), which was inhibited by SPARC siRNA. Knockdown of ILK attenuated extracellular H2O2 generation in TGF-β-stimulated fibroblasts. Our results indicated that SPARC is upregulated by TGF-β and is required for TGF-β-induced H2O2 production via activation of ILK, and this H2O2 production from fibroblasts is capable of causing epithelial cell injury.ConclusionsThe results presented in this study suggest that SPARC plays a role in epithelial damage in the IPF lung via enhanced H2O2 production from fibroblasts activated by TGF-β. Therefore, SPARC inhibition may prevent epithelial injury in IPF lung and represent a potential therapeutic approach for IPF.

Epigenetic Regulation of miR-17∼92 Contributes to the Pathogenesis of Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a disease of progressive lung fibrosis with a high mortality rate. In organ repair and remodeling, epigenetic events are important. MicroRNAs (miRNAs) regulate gene expression post-transcriptionally and can target epigenetic molecules important in DNA methylation. The miR-17~92 miRNA cluster is critical for lung development and lung epithelial cell homeostasis and is predicted to target fibrotic genes and DNA methyltransferase (DNMT)-1 expression. We investigated the miR-17~92 cluster expression and its role in regulating DNA methylation events in IPF lung tissue. Expression and DNA methylation patterns of miR-17~92 were determined in human IPF lung tissue and fibroblasts and fibrotic mouse lung tissue. The relationship between the miR-17~92 cluster and DNMT-1 expression was examined in vitro. Using a murine model of pulmonary fibrosis, we examined the therapeutic potential of the demethylating agent, 5'-aza-2'-deoxycytidine. Compared with control samples, miR-17~92 expression was reduced in lung biopsies and lung fibroblasts from patients with IPF, whereas DNMT-1 expression and methylation of the miR-17~92 promoter was increased. Several miRNAs from the miR-17~92 cluster targeted DNMT-1 expression resulting in a negative feedback loop. Similarly, miR-17~92 expression was reduced in the lungs of bleomycin-treated mice. Treatment with 5'-aza-2'-deoxycytidine in a murine bleomycin-induced pulmonary fibrosis model reduced fibrotic gene and DNMT-1 expression, enhanced miR-17~92 cluster expression, and attenuated pulmonary fibrosis. This study provides insight into the pathobiology of IPF and identifies a novel epigenetic feedback loop between miR-17~92 and DNMT-1 in lung fibrosis.

Antifibrotic effects of CXCR4 antagonist in bleomycin-induced pulmonary fibrosis in mice

Circulating fibrocytes had been reported to migrate into the injured lungs, and contribute to fibrogenesis via chemokine-chemokine receptor systems including CXCL12-CXCR4 axis. Here we hypothesized that blockade of CXCR4 might inhibit the migration of fibrocytes to the injured lungs and the subsequent pulmonary fibrosis. To explore the antifibrotic effects of blockade of CXCR4, we used a specific antagonist for CXCR4, AMD3100, in bleomycin-induced pulmonary fibrosis model in mice. Administration of AMD3100 significantly improved the loss of body weight of mice treated with bleomycin, and inhibited the fibrotic lesion in subpleural areas of the lungs. The quantitative analysis demonstrated that treatment with AMD3100 reduced the collagen content and fibrotic score (Aschcroft score) in the lungs. Although AMD3100 did not affect cell classification in bronchoalveolar lavage fluid on day 7, the percentage of lymphocytes was reduced by AMD3100 on day 14. AMD3100 directly inhibited the migration of human fibrocytes in response to CXCL12 in vitro, and reduced the trafficking of fibrocytes into the lungs treated with bleocmycin in vivo. These results suggest that the blockade of CXCR4 might be useful strategy for therapy of patients with pulmonary fibrosis via inhibiting the migration of circulating fibrocytes.

Local delivery of biodegradable pirfenidone nanoparticles ameliorates bleomycin-induced pulmonary fibrosis in mice

Our purpose was to assess sustained delivery and enhanced efficacy of pirfenidone-loaded nanoparticles after intratracheal instillation. Poly(lactide-co-glycolide) nanoparticles containing pirfenidone (NPs) were prepared and characterized. Biodistribution of NPs and solution was assessed using LC-MS after intratracheal administration in C57Bl/6 mice at 3 and 24 h and 1 week post-administration. Efficacy was tested in C57Bl/6 mice in a bleomycin-induced pulmonary fibrosis model. Mice received 10 μg pirfenidone intratracheally in solution or NPs, once a week, for 3 weeks after bleomycin administration. Drug effects were monitored on day 28. Lung hydroxyproline content, total number of cells, and numbers of macrophages, lymphocytes, and neutrophils in bronchoalveolar lavage (BAL) were assessed. Numbers of macrophages, lymphocytes, and neutrophils were assessed in the lung as well. NPs sustained significantly higher levels of pirfenidone in the lungs and BAL at 24 h and 1 week, compared to the solution group. Pirfenidone solution and NPs significantly reduced hydroxyproline levels by 57 and 81%, respectively, compared to bleomycin alone. At the end of 4 weeks, BAL cellularity was reduced by 25.4% and 56% with solution and NP treatment, respectively. The numbers of lymphocytes and neutrophils in the BAL were also reduced by 58.9 and 82.4% for solution and 74.5% and 89.7% for NPs, respectively. The number of inflammatory macrophages in the lung was reduced by 62.8% and the number of neutrophils was reduced by 59.1% in the NP group and by 37.7% and 44.5%, respectively, in the solution group, compared to bleomycin alone. In conclusion, nanoparticles sustain lung pirfenidone delivery and enhance its anti-fibrotic efficacy.

Fluorofenidone Attenuates Bleomycin-Induced Pulmonary Inflammation and Fibrosis in Mice Via Restoring Caveolin 1 Expression and Inhibiting Mitogen-Activated Protein Kinase Signaling Pathway

Idiopathic pulmonary fibrosis is a progressive, life-threatening, interstitial lung disease with no effective therapy. In this study, we evaluated the effects of fluorofenidone (FD), a novel pyridone agent, on a murine model of bleomycin-induced pulmonary inflammation and fibrosis. Institute for Cancer Research mice were intravenously injected with BLM or saline for 14 consecutive days. Fluorofenidone, pirfenidone (500 mg · kg · d, respectively), or vehicle was administered throughout the course of the experiment. Animals were killed on day 28, and various parameters reflecting pulmonary vascular permeability, influx of inflammatory cells, and levels of transforming growth factor β in the bronchoalveolar lavage fluid were assessed. Collagen I, α-smooth muscle actin, and fibronectin were measured by real-time reverse transcriptase-polymerase chain reaction or Western blot. Furthermore, caveolin 1 and activation of P38, extracellular signal-regulated kinase, and c-Jun N-terminal kinase were detected by Western blot. Fluorofenidone treatment significantly attenuated the increased pulmonary damage index score, the levels of proteins, transforming growth factor β, and the influx of cells in bronchoalveolar lavage fluid. Fluorofenidone also markedly reduced the expression of fibronectin, α-smooth muscle actin, and collagen I in mouse lung tissues. Inversely, FD restored caveolin 1 protein and mRNA expression, which was significantly downregulated in BLM-induced lung fibrosis. Fluorofenidone also inhibited phosphorylation of extracellular signal-regulated kinase, P38, and c-Jun N-terminal kinase. These findings collectively suggest that FD is an effective agent with antifibrotic and anti-inflammatory properties, and the mechanisms of its antifibrotic effect include regulating caveolin 1 expression and blocking mitogen-activated protein kinase signaling pathways.